This invention relates generally to the field of small conductance calcium activated potassium channels, and more specifically to the diagnosis, study, prevention and treatment of disorders related to these channels.
Action potentials in vertebrate neurons are followed by an after hyperpolarization (AHP) that may persist for several seconds and may have profound consequences for the neuronal firing pattern. The AHP has several components, which are distinct are mediated by different calcium activated potassium channels. Small conductance calcium activated potassium channels (SKCa, where xe2x80x9cSxe2x80x9d represents xe2x80x9csmallxe2x80x9d) underlie slow components of the AHP, which are responsible for spike-frequency adaption (Hotson, J. R., and Prince, D. A., J. Neurophysiol. 43:409, 1980). Small conductance channels have a unitary conductance of 4-14ps, are exquisitely sensitive to internal Ca2+, lack the property of voltage dependence and are blocked by nanomolar concentrations of the natural toxins apamin and scyllatoxin. These channels modulate the firing pattern of neurons via the generation of slow membrane afterhyperpolarizations (Nicoll, R. A., Science 241:545-551, 1988). The intermediate channels (IKCa, where xe2x80x9cIxe2x80x9d represents intermediate conductance) have a conductance of 11-40 pS, are blocked by charybdotoxin and clotrimazol, and are as sensitive to internal Ca2+ as SKCa channels (Ishii, T. M., et al., Proc. Natl. Acad. Sci. USA 94:11651-11656, 1997; Joiner, W. J., et al., Proc. Natl. Acad Sci. USA 94:11013-11018, 1997).
Channel dysregulation, particularly of the glutamate receptor/channels, figures prominently in pathogenic mechanisms involved in many processes leading to neurodegeneration, such as stroke, head trauma, and specific toxic drug reactions. Glutamate is the major excitatory neurotransmitter in the CNS, and while there are five known types of glutamate receptors, the NMDA-type ionotropic receptor/channel complex plays the most prominent role (Sucher, N. J., et al., Trends in Pharmacological Sciences 17:348-55, 1996; Kaku, D. A., et al., Science 260:1515-8, 1993). Overstimulation of the NMDA pathway produces calcium overload and the excitotoxic death of neurons (Choi, D. W., Neuron 1:623-34, 1988). The toxicity may arise through calcium-dependent proteases or through excessive signaling, perhaps leading to apoptosis. Extracellular Mg2+ produces tonic block of the NMDA receptor/channel complex, and in the presence of its agonist, the receptor/channel conducts an inward sodium and calcium current only if the membrane potential is concurrently being depolarized (Choi, 1988, supra). (This paried input paradigm perhaps also forms an elementary unit in learning, another function subserved by this receptor/channel.) As the cytosolic calcium concentration rises, SKCa channels are activated, generating slow membrane afterhyperpolarizations (Nicoll, R. A., Science 241:545-551, 1988; Johnson, S. W., and Seutin, V., Neuroscience Letters 231:13-16, 1997). NMDA current is physiologically terminated by this membrane repolarization, which restores the magnesium block. Since SKCa channels regulate ion flow through the NMDA-receptor/channel complex, they play a critical role in dampening neural excitability, and may also contribute to the development of neurological diseases associated with functional alterations in the NMDA signaling pathway (Johnson and Seutin, 1997, supra).
The SKCa are currents that have been described in a wide range of tissues, including brain (Lancaster, B. and Nicoll, R. A., J. Physiol. 389:187-203, 1987), peripheral neurons (Goh, J. W., and Pennefather, P. S., J. Physiol. 394:315-330,1987), skeletal muscle (Romey, G., and Lazdunski, M., Biochem. Biophys. Res. Commun. 118:669-674, 1984) adrenal chromaffin cells (Neely, A., and Lingle, C. J., J. Physiol. 452:97-131, 1992), leukocytes (Grissmer, S., et al., J. Gen. Physiol. 99:63-84, 1992), erythrocytes (Hamill, O. P., J. Physiol. 319:97P-98P, 1981), colon (Lomax, R. B., et al., Gut 38:243-247, 1996), and airway epithelia (Welsh, M. J., and McCann, J. D., Proc. Natl. Acad. Sci. USA 82:8823-8826, 1985). Certain types of SKCa channels have been distinguished by their sensitivities to the bee venom apamin, whereas other functionally related conductances appear insensitive (Sah, P., and AcLachlan, E. M., 1992, J. Neurophysiol. 74:1772-1776). The distinguishing features of the SKCa channels from the maxi-K calcium activated (BK) potassium channels are the SKCa channels"" low conductance (less than 50 pS), the weak or negligible dependence of their activity on membrane voltage, and their high affinity for calcium (EC50 less than 1 xcexcM) (e.g., Lancaster, B., and Zucker, R. S., J. Physiol. 475:229-239, 1994).
Recently, Imbert et al. reported the cloning of six novel cDNAs, each containing one or more long CAG repeats. These cDNAs were isolated from a lymphoblastoid cell cDNA expression library generated from patients with autosomal dominant cerebellar ataxia with a monoclonal antibody specific for polyglutamine sequences (Imbert et al., Nature Genetics 14:285, 1996). One of the cDNAs, designated AAD14, encoded the partial sequence of a putative protein of 228 amino acids in length. This polypeptide of unknown function contained two long polyglutamine stretches.
Kohler et al. first described a rat small conductance calcium activated potassium channel (rSKCa3) gene, and published the truncated form of the gene (Kohler et al., Science 274:1709, 1996). An alignment of the known members of the small conductance calcium-activated potassium channels was described by Joiner et al. (Proc. Natl. Acad Sci. 94:11013, 1997), who cloned the hSK4 gene, using the potassium channel signature sequence (TXXTXGYG, SEQ ID NO:9). Ishii et al. (Proc. Natl. Acad. Sci. USA 94:11651-6, 1997) further cloned an intermediate conductance calcium-activated potassium channel (h1K1).
Schizophrenia is a chronic disabling disorder with a lifetime morbid risk of 1% in the general population. The illness has a significant genetic component (Kendler, K. S., In: Relatives at Risk for Mental Disorders, Dunner, D. L., Gershon, E. S., and Barrett, J. E. (eds), Raven Press, New York, pp. 247-263, 1988) and often develops in early adulthood. The disease is characterized by a constellation of symptoms including hallucinations and delusions, disordered thinking and concentration, inappropriate emotional responses, catatonia, erratic behavior, and social and occupational deterioration. Although still controversial, xe2x80x9canticipationxe2x80x9d (an increase in severity through successive generations) has been found in studies of affected families (Bassett, A. S., and Honer, W. G., Am. J. Hum. Genet., 54:864-870, 1994). Trinucleotide repeat expansions have been found to underlie several Mendelian hereditary neurological diseases (Ashley, C. T., Jr., and Warren, S. T., Annu. Rev. Genet., 29:703-728, 1995; Timchenko, L. T., and Caskey, C. T., FASEB J., 10:1589-1597, 1996; among others).
Several human hereditary neurological diseases, such as Huntington""s disease, fragile X syndrome, myotonic dystrophy, spinal and bulbar muscular atrophy, Machado-Joseph disease, Friedrich""s disease and spinocerebellar ataxia, are associated with expanded trinucleotide repeats (typically greater than 35) within the coding region (e.g., Timchenko, L. T., and Caskey, C. T., FASEB J. 10:1589-97,1996; Hannan, A. J., J. Clin. Exp. Pharm. Physiol. 23:1015-20, 1996; Bates, G., Bioessays 18:1 75-8, 1996), untranslated sequences (e.g., Warren, S. T., and Ashley, C. T., Ann. Rev. Neurosci. 18:77-99, 1995; Tsilfidis, C., et al., Nature Genet. 1:192-195, 1992), or introns (Campuzano, V., et al., Science 271:1423-7, 1996) of genes. An association has been shown between the presence of anonymous CAG/CTG repeats and the development of schizophrenia and bipolar disease (O""Donovan, M. C., et al., Nature Genetics 10:380-1, 1995; O""Donovan et al., Psychological Med. 26:1145-1153, 1996; Cardino, A. G., et al., Brit. J. Psychiatry 169:766-771, 1996).
Intergenerational lengthening of such repeats is thought to underlie the phenomenon of xe2x80x9canticipationxe2x80x9d (Ashley and Warren, 1995, supra) observed in schizophrenia and bipolar disease, wherein the disease worsens with subsequent generations (Johnson, J. E., et al., Amer. J. Med. Genet. 75:275-280, 1997; Lipp, O., et al., Psychiatric Genet. 5:S8, 1995; Serbanescu-Grigorow, M., et al., Psychiatric Genet. 5:S10, 1995). The subset of these mutations caused by expansions of the CAG trinucleotide have been found only within the coding region of genes, and the encoded polyglutamine arrays may occur in different places within the open-reading-frame (Housman, D., Nature Genet. 10:3-4., 1995). In the past few years, expanded xe2x80x9canonymousxe2x80x9d CAG repeats in unidentified genes have been reported in patients with schizophrenia, with considerable overlap between allele distributions in patients and controls (O""Donovan, M. C., et al., Nature Genetics 10:380-381,1995; Morris, A. G., et al., Hum. Mol. Genet., 4:1957-1961, 1995; O""Donovan, M. C., et al., Psychol. Med. 26:1145-1153, 1996; O""Donovan, M. C., and Owen, M. J., Psychol. Med. 26:1-6, 1996; O""Donovan, M. C., and Owen, M. J., Annals Med. 28:541-546, 1996a).
The present invention is based on the discovery and cloning of the human small conductance calcium activated potassium channel type 3 (hKCa3/KCNN3) gene, which is expressed in neuronal cells, skeletal muscle, heart, and lymphocytes. Alterations in the hKCa3/KCNN3 gene or its protein product may enhance susceptibility to schizophrenia and/or bipolar disorder. Activators of this channel may be useful as neuroprotective agents (e.g., for preventing stroke or reducing neuronal injury following head trauma) via the xe2x80x9cphysiologicalxe2x80x9d inactivation of NMDA-receptor-mediated excitotoxic mechanisms. In addition, since hKCa3/KCNN3 is expressed in lymphocytes, the channel may play a role in modulating calcium-signaling during the immune response. The gene is expressed in skeletal muscle and the heart where it may play a similar role in modulating signaling. Thus hKCa3/KCNN3 may be involved in neuropsychiatric, neurological, neuromuscular, and immunological disorders.
In a first embodiment, substantially purified hKCa3/KCNN3 polypeptide and isolated polynucleotides encoding hKCa3/KCNN3 polypeptides are provided. Antibodies which bind to hKCa3/KCNN3 polypeptides are also disclosed.
In another embodiment, a method for identifying a compound which affects hKCa3/KCNN3 polynucleotide or polypeptide is provided. Such compounds include drugs and small molecules.
A method for diagnosis of a subject having or at risk of having a hKCa3/KCNN3-associated disorder is also provided. A method of determining the prognosis of a subject with an hKCa3/KCNN3-associated disorder is disclosed.
In another embodiment, a method of treating a subject having or at risk of having an hKCa3/KCNN3-associated disorder by administering a therapeutically effective amount of a polynucleotide encoding SEQ ID NO:2 is also provided. A method of treating a patient having or at risk of having an hKCa3/KCNN3-associated disorder by introducing into a cell of the patient a nucleotide sequence encoding SEQ ID NO:2 under the control of a eukaryotic promoter so that the cell expresses SEQ ID NO:2 is provided.
A composition for administration of hKCa3/KCNN3 to a patient of a therapeutically effective amount of hKCa3/KCNN3 polypeptide is disclosed.
In an additional embodiment, a kit useful for detecting the presence of hKCa3/KCNN3 polypeptide in a sample from a subject having a hKCa3/KCNN3-associated disorder is provided. A kit useful for the detection of polynucleotide encoding hKCa3/KCNN3 in a subject having a hKCa3/KCNN3 associated disorder is also provided.
In a further embodiment, transgenic nonhuman animals having a transgene encoding hKCa3/KCNN3 are also described.